Project Summary/Abstract Plasticity of gene expression programs allows cells to respond and adapt to stress. While adaptation involves both transcriptional and post-transcriptional changes, translational regulation is particularly crucial in situations that demand a rapid response. Cells respond to stress such as hypoxia, starvation, oxidative stress, heat shock or viral infections by shutting down translation and sequestering mRNA into stress granules; dynamic, non-membrane bound, phase-separated cytoplasmic organelles. The ability of cells to form stress granules is particularly important in the ability of cancer cells to survive chemotherapeutic treatments. However, stress granules are not monotypic. G3BP, a core nucleator protein of stress granules, has at least three known variants - G3BP1, G3BP2a and G3BP2b. G3BP variants can form homo or hetero-multimers in the context of stress granules, and have also been shown to have different tissue specificities. However, the functional consequences of this variation amongst granule nucleating proteins has not been studied. The absence of this knowledge renders our understanding about the cellular response to various insults incomplete. G3BP variants also have differential granule-forming capacities, indicating more than one dimension of heterogeneity amongst cellular stress granules. Further, our data suggests that G3BP variants are unable to compensate for each other functionally, indicating divergence in the stress response. Therefore, we hypothesize that the heterogeneity in stress granule composition is important for the cell to be able to respond and adapt to diverse stresses, and that granules of different compositions have different effects on the mRNA fates in response to stress. We will address this hypothesis via the following specific aims: (1) Identify role of stress granule heterogeneity in cellular resistance to exogenous stresses: We will subject cells that can produce only a limited number of or only a single granule subtype(s) to a wide panel of stressors and monitor absolute and relative fitness using a sensitive flow cytometry assay and (2) Elucidate the mechanism(s) of granule-dependent differential fitness in response to stressors: Using a combination of molecular biology and high-throughput sequencing technologies, we will measure localization of transcripts to granules nucleated by specific G3BP variants. A recently developed optogenetic tool will allow us to induce granule formation in the absence of stress, thereby independently measuring the effect of granules on cytoplasmic translation. My goal is to become a principal investigator pursuing an independent research program focused on deciphering cellular responses to stress. UCSF is an ideal environment to pursue an interdisciplinary approach to answering these fundamental biological questions. Completion of the proposed work will expand our knowledge of the mechanisms underlying the cellular response to stress, informing on responses to pathologies and therapeutic regimens.